U.S. Pat. Nos. 4,683,202 and 4,683,195 describe a process for amplifying and detecting nucleic acid sequences in a process known as the Polymerase Chain Reaction, or PCR. The PCR process consists of three basic steps: 1) denaturation of the template strands at elevated temperature; 2) annealing at hybridization temperature of oligonucleotide primers to the template DNA at the 3' ends of the sequence of interest; and 3) extension by a thermostable DNA polymerase in the presence of nucleotide triphosphates of the 3' ends of the primer to replicate the desired template sequence. Steps 1-3 are repeated in cyclic fashion so that the primer extension products of any cycle become the templates for replication in subsequent cycles and the target sequence is amplified exponentially.
U.S. Pat. No. 4,683,202 also claims a staged method of PCR in which a second set of primers is used to amplify a smaller DNA sequence contained within the DNA sequence amplified by the first primer set. The method, commonly referred to as nested PCR, is recognized as a more sensitive and specific method.
The optimization of PCR is considered in a number of publications (PCR Protocols, A Guide to Methods and Applications, Innis M. A., Gelfand, D. H., Sninsky, J. J, and White, T. J. eds., Academic, N.Y., 1990; Linz, U, Delling, U, and Rubsamen-Waigmann, H., J. Clin. Chem. Clin. Blochem., 28, 5, 1990; Rychlik, W., Spencer, W. J., and Rhoads, R. E., Nucl. Acids Res., 18, 6409, 1990; Wu, D. Y., Uggozoli, L., Pal, B. K., Qian, J, and Wallace, R. B., DNA Cell Biol., 10, 233, 1991) . Guidance is given in the selection of buffer, magnesium, nucleotide triphosphate, primer, and DNA polymerase concentrations as well as the times and temperatures employed during cycling. Particular emphasis is placed on the optimal choice of primer sequence to avoid regions of high secondary structure or complementarity between primers. Emphasis is also placed on the optimal choice of the primer annealing temperature to maximize the yield and selectivity of the amplification. However, descriptions of the influence of primer concentration and annealing time on the selectivity and yield of the amplification are not provided.
Nested PCR has been shown to increase the sensitivity of target DNA detection by at least two orders of magnitude while minimizing background from non-target DNA amplification (Garson, J. A., Tedder, R. S., Briggs, M., Tuke, P., Glazebrook, J. A., Trute, A., Parker, D., Barbara, J. A., Contreras, M., and Aloysius, S., Lancet, 335, 1419, 1990; Porter-Jordan, K., Rosenberg, E. I., Keiser, J. F., Gross, J. D., Ross, A. M., Nasim, S., and Garrett, C. T., J. Med. Virol., 30, 85, 1990). For effective nested amplification, it is necessary to terminate the amplification of the outer primer set after the first stage to allow the inner primers alone to amplify the DNA in the second stage. To minimize the carryover of outer primer into the second stage, the first stage product has traditionally been diluted (Rimstad, E., Hornes, E., Olsvik, O., and Hyllseth, B., J. Clin. Microbiol., 28, 2275, 1990) or only a small fraction (2-10%) of it is added to the second stage reaction (Welch, D., Lee, C. H., and Larsen, S. H., Appl. Env. Microbiol, 56, 2494, 1990).
Gyllensten, U. B., and Erlich, H. A., Proc. Natl. Acad. Sci., 85, 7652, 1988, describe asymmetric amplification in which one of the primers of a pair is present in one-fiftieth or one-hundredth of the usual concentration. By carrying out enough cycles, the primer present at the lower concentration will become depleted, and thus the DNA generated from the remaining primer will be selectively enriched in subsequent cycles. The primer depletion method has the disadvantage that the number of cycles required to deplete the first stage primers depends on the template DNA concentration initially present. In situations where there is an initially low sample DNA concentration the number of cycles required can be quite large (30-40), and the method is therefore not optimal for a nesting approach which depends on a lower (20-25) number of cycles per stage.
Igarashi et al., EPA Publication No. 0469610A1, claim an assay wherein a reduced primer concentration in the first stage of nested PCR gives rise to a superior target to background amplification. A reduced first stage primer concentration is an integral part of the kinetically controlled methods described in this invention. However, Igarashi's nesting protocol differs from the instant method in at least three respects. 1) The instant method demonstrates that an increased annealing time in the first stage in coordination with the reduced primer concentration is essential for high efficiency of amplification in the first stage. Igarashi operates at a constant annealing time in both stages. 2) The instant method demonstrates that the primer concentration and the annealing time must be chosen on the basis of the annealing kinetics of each primer/template combination. Igarashi does not disclose a method of arriving at optimum amplification conditions to maximize the claimed improvement. 3) Igarashi uses only 10% of the product of the first stage of amplification in the second stage, whereas the instant method utilizes the whole product of the first stage during the second stage.
Yourno, PCR Methods and Applications, 2, 60, 1992, describe a method of nested PCR in a single, closed amplification tube which is approximately 100 times more sensitive than single stage amplifications. In this method, the second stage primer and reaction mix are sequestered from the first stage amplification by entrapment in high melt agarose in a cooled portion of the tube above the temperature cycled liquid. Prior to the second stage, tubes are centrifuged to drop the agarose into the temperature cycled portion, where the agarose melts and releases the second stage reagents. Yourno also operates with a several-fold reduced first stage primer concentration. Again, this disclosure teaches constant annealing time for the first and second stages; does not give consideration to the amplification efficiency of each primer in each stage; and does not disclose any kinetic model to manipulate the amplification conditions and thereby optimally practice the nesting method.
Erlich, H. A., Gelfand, D., and Sninski, J. J., Science, 252, 1643, 1991, describe a "drop-in, drop-out" nesting in which both primer pairs are present initially and no manipulation of the reaction mixture is needed during the course of amplification, minimizing the risk of sample cross-contamination. The outer primer set is longer, or has a higher GC content than the inner set. In addition, it is implied that the extension product of the outer primer set is substantially longer or has a higher GC content than the inner primer extension product. If sufficiently high annealing and denaturation temperatures are used in the first stage, inner primer annealing is prevented while outer primer annealing, extension and denaturation proceeds. The annealing and denaturation temperatures in the second stage are reduced to enable inner primer annealing and to prevent outer primer extension product denaturation. The inner primers are thus "dropped in" in the second stage by proceeding with a reduced annealing temperature, and outer primer amplification is a "dropped out" by the lower denaturation temperature. Alternatively, the outer primers can be "dropped out" by depleting them in the first stage in a manner analogous to asymmetric amplification. The general applicability and effectiveness of "dropping out" primers by reduced denaturation temperature is not disclosed.
The kinetics of annealing oligonucleotide probes to DNA and the reannealing of denatured strands of genomic DNA have been studied (Britten, R. J. and Kohne, D. E., Science, 161, 529, 1968, Wetmur, J. G., J Molec. Biol., 31, 329, 1968, Young, B. D. and Paul J., Blochem J., 135, 573, 1973) and described as second order. Such rate modelling has contemplated hybridization efficiency as a function of DNA concentration and contact time in studies involving affinity capture for detection purposes (Wood, T. G., and Lingrel, J. B., J. Biol. Chem., 252, 457, 1977; and Mc Mahon, M. E., European Patent Application 90104413.1). However, primer annealing kinetics has not been a variable which has been recognized in the design and control of gene amplification by PCR.
The present invention is a method to perform nested PCR in which the entire product of the first stage of nesting is used in the second stage without the need to dilute, deplete, or otherwise remove the outer primers. Applicants have achieved efficient selective amplification of the outer primers in the first stage, and their dropout in the second stage, solely by controlling the rate of primer annealing to the template at each stage. The annealing kinetics have been manipulated by carefully selecting and controlling primer concentrations and annealing times in the first and second stages according to predictions of Applicants' second order kinetic model wherein the parameters are evaluated independently for each primer pair and template.
Applicants' kinetically controlled method of dropping out the outer primers is distinct and advantageous over the existing art in that 1) primer annealing temperature need not be varied throughout the nesting stages; 2) the second stage can be activated after any desired number of cycles; and 3) the method is independent of the starting nucleic acid concentration and also of the relative sizes of the primer extension products. The method can be practiced within one reaction vessel.
Although it is contemplated that Applicants' abovedescribed nested amplification method may be utilized in any procedure wherein specific segments of nucleic acids are replicated for analytical, diagnostic or genetic cloning purposes, the invention has been embodied in the instant application in a highly sensitive method for the identification of microbial contaminants in food. Specifically, Applicants' food diagnostic methodology entails in the first step, identifying a random, unique segment of DNA for each individual microorganism of interest which will be diagnostic for that microorganism. To identify and obtain this diagnostic nucleic acid segment, a series of polymorphic markers is generated from each organism of interest using single primer RAPD (Random Amplified Polymorphic DNA) analyses as described in Nucleic Acid Research, Vol. 18, No. 22, pp. 6531-35, Williams et al., and U.S. Pat. No. 5,126,239 (1992), E. I. du Pont de Nemours and Company. The RAPD series from each organism is compared to similarly-generated RAPD series from other organisms, and a RAPD marker unique to each organism of interest is selected. The unique markers are then isolated, amplified and sequenced. Outer primers and inner primers for each marker may then be developed. These primers will comprise sequence segments within the RAPD marker, and the inner set of primers will be complementary to the 3' ends of the target piece of nucleic acid. These outer and inner nested primers may then be used in Applicants' improved nested PCR amplification method, on food samples for example, to enable the highly sensitive, rapid and precise identification of microbial contaminants.
Other methods are known which utilize nested PCR techniques for the identification of microbial food contaminants. However, none of these methods employ Applicants' improved nested PCR which accomplishes highly efficient amplification of the diagnostic nucleic acid target by manipulation of primer concentration and annealing times at each stage of amplification. (Olive, M. D., J. Clin. Microbiol., 27, 261, 1989; Wilson, I. G., Cooper, J. E. and Gilmour, A., Appl. Env. Microbiol., 57, 1793, 1991; Furrer, B., Candrian, U., Hoefelein, C., and Luethy, J., J. Appl. Bact., 70, 372, 1991).